Biofilm formation and its induced biocorrosion of metals in seawater

BIOFILM FORMATION AND ITS INDUCED
BIOCORROSION OF METALS IN SEAWATER









SHENG XIAOXIA

NATIONAL UNIVERSITY OF SINGAPORE
2007







BIOFILM FORMATION AND ITS INDUCED
BIOCORROSION OF METALS IN SEAWATER









SHENG XIAOXIA
(B.ENG. (Hons.), ZHEJIANG UNIVERSITY)

A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF CHEMICAL AND BIOMOLECULAR
ENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE
2007

Acknowledgements
i
ACKNOWLEDGEMENTS
I first would like to express my deepest gratitude and appreciation to my
supervisor Prof. Ting Yen Peng, for his constant guidance and inspiration throughout
my graduate studies. It was his patience and support through the years which inspired
me to preserve in my quest. I also would like to thank my co-supervisor, Prof. Simo
Olavi Pehkonen, for providing extremely valuable discussions and suggestions
regarding my research. I am very grateful towards Dr. He Jianzhong for helping me
conduct the molecular biology experiments, and for her insightful discussions for
pointing out the directions to improve my research work.
This work has received a great deal of support and assistance from the lab
officers Ms. Li Fengmei, Ms. Li Xiang, Ms. Sylvia Wan, Mr. Qin Zhen, and Mr. Boey
Kok Hong for their assorted help around the lab. I would like to acknowledge Ms.
Samantha Fam for her guidance on the operation of AFM. I also thank Mr. Ng Kim
Poi for preparing the metal coupons and making the corrosion cell.
Special thanks to my friends Zhao Quangqiang, Zhu Zhen, Wang Yan, Xu
Tongjiang, and Xu Ran for their friendship. Their help in my life made my graduate
study an enjoyable and exciting experience.
I would like to show my greatest appreciation to my husband, Zhang Ning, and
my parents for their support and encouragement.
This work was supported from Tropical Marine Science Institute (Singapore)

National University of Singapore (Research Grant RP-279-000-173-112).
Table of Contents
ii
TABLE OF CONTENTS

ACKNOWLEDGEMENTS i
SUMMARY v
LIST OF FIGURES vii
LIST OF TABLES xi
NOMENCLATURE xii
CHAPTER 1 INTRODUCTION 1
1.1 Biofilm Formation on Metal Surfaces 3
1.2 Mechanisms of Biocorrosion 6
1.3 Bacteria Related to Biofilm Formation and Biocorrosion 7
1.3.1 Sulphate-reducing Bacteria (SRB) 7
1.3.2 Other Bacteria 10
1.4 Methods for the Inhibition of Biofilm and Biocorrosion 14
1.4.1 Layer-by-layer (LBL) Polyelectrolyte Multilayer Coating 14
1.4.2 Organic Inhibitors 17
1.5 Objectives and Scope of This Work 21
CHAPTER 2 MATERIALS AND METHODS 24
2.1 Metal Coupons 24
2.2 Microorganisms 24
2.3 Isolation and Identification of Strain SJI1 25
2.3.1 Morphological Characterization 25
2.3.2 Physiological Studies 26
2.3.3 16S rRNA Sequence Analysis 28
2.3.4 Phylogenetic Analysis 28
2.3.5 Nucleotide Sequence Accession Number 29
2.4 Biofilm Formation 29

2.4.1 Cell Immobilization 29
2.4.2 Zeta Potential (ζ) and Contact Angle Measurements 30
2.4.3 Confocal Laser Scanning Microscopy (CLSM) 31
2.4.4 AFM Operation of Force Measurement 31
2.5 Biofilm and Biocorrosion of Stainless Steel AISI 316 and Its Prevention 32
2.5.1 Biofilm and Biocorrosion Experiment Setup 32
2.5.2 Scanning Electron Microscopy (SEM) 33
2.5.3 Atomic Force Microscopy (AFM) 34
2.5.4 Electrochemical Impedance Spectroscopy (EIS) 34
2.6 Preparation of Layer-By-Layer (LBL) Coating 35
2.6.1 Polyelectrolyte Solutions 35
2.6.2 Layer-by-layer (LBL) Technique 36
Table of Contents
iii
2.6.3 Stability of the PEM on Functionalized SS316 37
CHAPTER 3 ISOLATION, CHARACTERIZATION AND IDENTIFICATION OF A
MARINE SULPHATE REDUCING BACTERIA 39
3.1 Cell Morphology 39
3.2 Growth of Desulfovibrio singaporenus Strain SJI1 on Lactate and Acetate 40
3.3 Physiological Properties 44
3.4 16S rRNA Gene Sequence and Phylogenetic Analysis 47
3.5 Summary 51
CHAPTER 4 BIOFILM FORMATION AND FORCE MEASUREMENT 52
4.1 Force Measurement in the Fluid 52
4.1.1 Typical Force Curves 52
4.1.2 Forces Between the Cell Tip and Different Metal Substrates 55
4.1.3 Cell Tip-Cell Lawn Interactions 60
4.1.4 Influence of Nutrient and Ionic Strength on the Cell-Metal Interaction
64
4.1.5 Influence of Solution pH on the Cell-Metal Interaction 68

4.2 Ex-situ Force Measurement 73
4.3 Summary 78
CHAPTER 5 SULPHATE REDUCING BACTERIA BIOFILM AND ITS INDUCED
BIOCORROSION OF STAINLESS STEEL AISI 316 80
5.1 AFM Image Analysis 80
5.1.1 Biofilm Investigation 80
5.1.2 Pits Investigation 84
5.2 EIS Results 88
5.2.1 Control Coupons in EASW 88
5.2.2 Coupons in EASW with D. desulfuricans 95
5.2.3 Coupons in EASW with D. singaporenus 97
5.2.4 Comparison of the Coupons with and without SRB 98
5.3 Summary 100
CHAPTER 6 BIOFILM AND BIOCORROSION INHIBITION USING
LAYER-BY-LAYER COATING 102
6.1 Surface Functionalization of SS316 and the Stability of the Multilayers 102
6.2 XPS Analysis of the Functionalized Stainless Steel 104
6.3 Biofilm Viability Study by CLSM 106
6.4 Biofilm and Biocorrosion Study Using AFM 108
6.5 Biocorrosion Study Using Linear Polarization Analysis 110

Table of Contents
iv
CHAPTER 7 BIOFILM AND BIOCORROSION INHIBITION USING AN
ORGANIC INHIBITOR 112
7.1 Evaluation of Organic Corrosion Inhibitor on Abiotic and Biotic Corrosion of
Mild Steel 112
7.1.1 XPS Analysis 112
7.1.2 Bacteria Concentration 114
7.1.3 EIS Analysis 115

7.1.4 Linear Polarization Analysis and Potentiodynamic Scanning Curves118
7.1.5 SEM Analysis 122
7.1.6 AFM Analysis 126
7.1.7 Adsorption Isotherm 128
7.2 Evaluation of Organic Corrosion Inhibitor on Abiotic and Biotic Corrosion of
SS316 130
7.2.1 EIS Analysis 130
7.2.2 Linear Polarization Analysis 133
7.2.3 CLSM Analysis 134
7.2.4 AFM Analysis 136
7.2.5 Adsorption Isotherm 138
7.3 Summary 139
CHAPTER 8 CONCLUSIONS AND RECOMMENDATIONS 141
8.1 Conclusions 141
8.2 Recommendations 146
REFERENCES 149


Summary
v
SUMMARY
Biocorrosion, also termed as microbiologically influenced corrosion (MIC),
refers to the electrochemical process where the participation of the microorganisms on
a metal surface accelerates the corrosion reaction on the metal surface. An important
step of biocorrosion process is the formation of a biofilm, a microbial community
which is enveloped by adhered extracellular biopolymer substances (EPS) these
microbial cells produce on the surface of a liquid and a surface. In this thesis, several
issues related to biofilm and biocorrosion on metals are addressed. These include: (i)
the isolation and characterization of a novel marine sulphate-reducing bacteria (SRB)
strain from local seawater, (ii) investigating bacteria-metal interactions, (iii)

investigating biofilm and its induced biocorrosion of two SRB strains on stainless
steel 316 (SS316), and (iv) biofilm and biocorrosion prevention using an organic
inhibitor and a layer-by-layer coating on the metal substrate.
A novel sulphate-reducing bacterium, designated Desulfovibrio singaporenus
strain SJI1, was isolated from seawater near St. John Island, Singapore. The isolate is
rod, curved-shaped and motile, and is a typical moderately halophilic and mesophilic
strain. Interestingly, D. singaporenus completely oxidizes lactate to acetate via
pyruvate as the intermediate during sulphate reduction. Acetate is further partially
oxidized to CO
2
when it is used as an electron donor.
The adhesion of two anaerobic sulphate-reducing bacteria (D. desulfuricans and
D. singaporenus) and an aerobe (Pseudomonas sp.) to four polished metal surfaces
(i.e. stainless steel AISI 316, mild steel, aluminum, and copper) was examined using a
force spectroscopy technique with an atomic force microscopy (AFM). Using a
modified bacterial tip, the attraction and repulsion forces (in the nano-Newton range)
between the bacterial cell and the metal surface in aqueous media were quantified.
Results show that the bacterial adhesion force to aluminum and to copper is the
highest and the lowest respectively among the metals investigated. The bacterial
adhesion forces to metals are influenced by the surface charges and the
hydrophobicity of the metal and bacteria. The cell-cell interactions show that there are
Summary
vi
strong electrostatic repulsion forces between bacterial cells.
Biocorrosion of SS316 by D. desulfuricans and D. singaporenus was
investigated. The biofilm and pit morphology that developed with time were analyzed
using atomic force microscopy (AFM). Electrochemical impedance spectroscopy (EIS)
results were interpreted with an equivalent circuit to model the physicoelectric
characteristics of the electrode/biofilm/solution interface. D. desulfuricans formed one
biofilm layer on the metal surface, while D. singaporenus formed two layers: a biofilm

layer and a ferrous sulfide deposit layer. AFM images corroborated results from the EIS
modeling which showed biofilm attachment and subsequent detachment over time.
These results indicate that SRB could directly react with metal surface, and it plays
direct role in the biocorrosion.
A layer-by-layer coating on SS316 substrate alternately with quaternized
polyethylenimine (q-PEI) and poly(acrylic)acid (PAA) to form polyelectrolyte
multilayers (PEM) was investigated. The PEM were stable in seawater. The
antibiocorrosion ability of PEM on stainless steel was assessed using Pseudomonas
sp., D. desulfuricans and D. singaporenus. Compared to the bare stainless steel, the
corrosion rates and the pit depths decreased for the PEM functionalized SS316.
Biofilm growth on the substrate was inhibited by the antibacterial effect of q-PEI as
shown by confocal laser scanning microscopy (CLSM). These results indicate that
PEM have potential applications in the inhibition of biocorrosion of metal substrates.
Corrosion inhibition of mild steel and SS316 by an organic inhibitor
2-Methylbenzimidazole (MBI) in seawater was also investigated using direct current
polarization, XPS, EIS, SEM, CLSM, and AFM. MBI was shown to be an effective
inhibitor in controlling abiotic corrosion as well as biocorrosion by D. desulfuricans
and D. singaporenus. Tafel plots revealed that MBI predominantly controls the
cathodic reaction. The corrosion inhibition effect of MBI on MIC is partially due to
the inhibition of the bacterial activity. The adsorption of MBI on the steel surface
follows a Langmuir adsorption isotherm model.

List of Figures
vii
LIST OF FIGURES

Figure 1.1 Structure of 2-Methyl-benzimidazole (MBI) 20
Figure 2.1 Derivatization of q-PEI 36
Figure 2.2 Layer-by-layer (LBL) coating of q-PEI and PAA multilayer on polished
SS316 37

Figure 3.1 Images of strain SJI1 on a SS316 coupon: (a) a single cell (x10,000); (b)
cells growing on SS316 (x5,000); (c) an AFM phase image of an individual
cell with a single polar flagellum (scale 4 μm × 4 μm) 40
Figure 3.2 (a) Time course of the growth of strain SJI1 showing increase in cell density
(♦) and decrease in sulphate concentration (►); (b) The consumption of
lactate (▲) and the production of acetate (●) and pyruvate (■)
accompanying bacterial growth. Error bars indicate standard deviation,
which are not shown when they are smaller than the symbol 42
Figure 3.3 Nucleotide sequence of the 16S rRNA gene of strain SJI1 (deposited in the
Genbank database on 16
th
April 2007 under accession number EF178280).
48
Figure 3.4 A phylogenetic tree based on 16S rRNA gene sequences showing the
position of strain SJI1 within the genus Desulfovibrio and in relation to
other sulphate-reducing bacteria. The tree was calculated using the
neighbor-joining method. Bar, 2% sequence divergence 49
Figure 4.1 A scanning electron microscope image of a silicon nitride tip coated with
Pseudomonas sp 52
Figure 4.2 A typical force-distance curve between a Pseudomonas sp. coated tip and
SS316 54
Figure 4.3 Force-distance curves when a Pseudomonas sp. cells coated tip was (a)
extended to and (b) retracted from different metal substrates in artificial
seawater 58
Figure 4.4 Force-distance curves when a D. desulfuricans cells coated tip was (a)
extended to and (b) retracted from different metal substrates in artificial
seawater 58
Figure 4.5 Force-distance curves when a D. singaporenus cells coated tip was (a)
extended to and (b) retracted from different metal substrates in artificial
seawater 59

Figure 4.6 CLSM images of Pseudomonas sp. adhering onto (a) mild steel, (b) copper,
(c) aluminum, and (d) on SS316 in artificial seawater. The scale bar is 500
μm for all images 60
List of Figures
viii
Figure 4.7 Force-distance curves when bacteria coated tip was extended to the substrate
in artificial seawater: (a) D. singaporenus, (b) Pseudomonas sp., and (c) D.
desulfuricans 63
Figure 4.8 Force-distance curves when a cells-coated tip was retracted from SS316 in
different solutions (a) Pseudomonas sp.; (b) D. desulfuricans; (c) D.
singaporenus 66
Figure 4.9 CLSM images of Pseudomonas sp. adhering onto SS316 in (a) DIW; (b)
ASW; (c) EASW 68
Figure 4.10 The adhesion force between cell probe and SS316 in ASW with various pH:
(a) Pseudomonas sp.; (b) D. desulfuricans; (c) D. singaporenus 71
Figure 4.11 XPS measurement of Fe 2p spectra in ASW at various pH: (a) pH 3, (b) pH
5, (c) pH 7, and (d) pH 9 72
Figure 4.12 A contact mode AFM image of a biofilm on SS316 76
Figure 4.13 Force measurements on the biofilm surface with D. singaporenus: (A—on
cell, B—at cell periphery, C—on biofilm substrate, D—on deposit and
E—at deposit periphery) 77
Figure 4.14 Force measurements on the biofilm surface with D. desulfuricans: (A—on
cell, B—at cell periphery, C—on biofilm substrate, D—on deposit and
E—at deposit periphery) 77
Figure 5.1 Atomic Force Microscopy images of stainless steel AISI 316 coupons with
D. desulfuricans biofilm; (a) 4-day-immersion; (b) 14-day-immersion; (c)
24-day-immersion; (d) 34-day-immersion; (e) 44-day-immersion 82
Figure 5.2 Atomic Force microscopy images of SS316 coupons with D. singaporenus
biofilm; (a) 4-day-immersion; (b) 14-day- immersion; (c) 24-day-
immersion; (d) 34-day- immersion; (e) 44-day- immersion 83

Figure 5.3 Two- and three-dimensional images of (a) a single pit, and (b) a D.
desulfuricans cell on the SS316 coupons 85
Figure 5.4 Section analysis on the SS316 coupons: (a) height profile of D. desulfuricans
cells; (b) depth profile of a small pit; (c) depth profile of a large pit 86
Figure 5.5 Depth of pits on SS316 at different time of exposure 87
Figure 5.6 SEM images for biofilm on the SS316 in MASW with (a) D. desulfuricans
and (b) D. singaporenus 87
Figure 5.7 EIS analysis for the samples at 35
th
day of immersion: (a) control coupon; (b)
coupon with D. desulfuricans; (c) coupon with D. singaporenus 90
Figure 5.8 Equivalent Circuit models: (a) Model of R(Q[R(QR)]) for control coupons;
(b) Model of R(Q[R(QR)(QR)]) for control coupons; (c) Model of
R(Q[R(QR)(QR)]) for coupons in EASW with D. desulfuricans; (d) Model
List of Figures
ix
of R(Q[R(QR)(QR)(QR)]) for coupons in EASW with D. singaporenus.92
Figure 5.9 Experimental EIS data (symbol) and their fitted data (line) for (a) a SS316
coupon; (b) coupon with D. desulfuricans; (c) coupon with D. singaporenus.
93
Figure 5.10 Cyclic polarization curves of SS316 exposed to EASW for (a) 7 days; (b)
14 days; (c) 21 days. (d) Potentiodynamic scanning curve of SS316 coupon
exposed to EASW with D. desulfuricans for 7 days 95
Figure 6.1 Contact angle measurements for the different layers of coating 103
Figure 6.2 The stability test of the functionalized SS316 in EASW 104
Figure 6.3 XPS wide scan for (a) the pristine SS316 and (b) q-PEI/PAA multibilayers of
the functionalized SS316 105
Figure 6.4 N 1s spectra for (a) the pristine SS316 and (b) q-PEI/PAA multibilayers of
the functionalized SS316 106
Figure 6.5 CLSM images for the biofilm on (1) the pristine, and (2) the functionalized

SS316 in EASW for 5 weeks with (a) Pseudomonas sp., (b) D.
desulfuricans, and (c) D. singaporenus 107
Figure 6.6 AFM surface roughness analysis for the biofilm on (a) the pristine SS316,
and (b) the functionalized SS316 after immersing in EASW for 1, 3, and 5
weeks 108
Figure 6.7 AFM bearing analysis for pit volume formed on (a) the pristine SS316, and
(b) the functionalized SS316 after immersing in EASW for 1, 3, and 5
weeks 109
Figure 7.1 N 1s spectra for (a) the pristine mild steel; (b) the mild steel deposited with
MBI 113
Figure 7.2 Fe 2p spectra for (a) the pristine mild steel; (b) the mild steel deposited with
MBI 114
Figure 7.3 Nyquist plots for mild steel in EASW for 24 hours (a) without bacteria; (b)
with D. singaporenus; (c) with D. desulfuricans 117
Figure 7.4 Equivalent circuit for the metal/liquid interface 117
Figure 7.5 Tafel polarization curves of pristine mild steel and inhibited mild steel in
EASW for 24 hours (a) without bacteria; (b) with D. desulfuricans; (c) with
D. singaporenus 119
Figure 7.6 Potentiodynamic scanning curves of mild steel exposed to EASW for 24
hours (a) without bacteria; (b) with D. desulfuricans; (c) with D.
singaporenus 122
Figure 7.7 SEM images of mild steel in EASW for 24 hours (a) without MBI; (b) with
List of Figures
x
MBI at 0.1 mM; (c) with MBI at 0.5 mM; (d) with MBI at 1 mM.
(magnification x1,000) 123
Figure 7.8 SEM images of mild steel in EASW with D. desulfuricans for 24 hours (a)
without MBI; (b) with MBI at 1 mM; (c) with MBI at 2.5 mM.
(magnification x1,000) 124
Figure 7.9 SEM images of mild steel in EASW with D. singaporenus for 24 hours (a)

without MBI; (b) with MBI at 1 mM; (c) with MBI at 2.5 mM.
(magnification x1,000) 124
Figure 7.10 Biofilm on mild steel (a) D. singaporenus without MBI; (b) D.
singaporenus with MBI at 1 mM; (c) D. desulfuricans without MBI; (d) D.
desulfuricans with MBI at 1 mM 125
Figure 7.11 AFM images of mild steel in EASW with D. desulfuricans for 24 hours (a)
without MBI; (b) with MBI at 1 mM; (c) with MBI at 2.5 mM 127
Figure 7.12 AFM images of mild steel in EASW with D. singaporenus for 24 hours (a)
without MBI; (b) with MBI at 1 mM; (c) with MBI at 2.5 mM 127
Figure 7.13 The application of the Langmuir isotherm model to the corrosion protection
behavior of MBI to mild steel 130
Figure 7.14 Nyquist plots for SS316 in EASW for 1 week (a) without bacteria; (b) with
D. desulfuricans; (c) with D. singaporenus 132
Figure 7.15 CLSM images of SS316 in EASW (a) with D. desulfuricans; (b) with D.
desulfuricans + MBI (1 mM); (c) with D. desulfuricans + MBI (2.5 mM).
135
Figure 7.16 CLSM images of SS316 in EASW (a) with D. singaporenus; (b) with D.
singaporenus + MBI (1 mM); (c) with D. singaporenus + MBI (2.5 mM).
136
Figure 7.17 AFM images of SS316 in EASW (a) with D. desulfuricans, (b) with D.
desulfuricans + MBI 1 mM, (c) with D. singaporenus, (d) with D.
singaporenus + MBI 1 mM for 1 week 137
Figure 7.18 The application of the Langmuir isotherm model to the corrosion protection
behavior of MBI to SS316 139

List of Tables
xi
LIST OF TABLES
Table 3.1 Utilization of organic compounds in the presence of sulphate and
fermentation of carbon source in the absence of electron acceptor for

strain SJI1 46
Table 3.2 Comparison of strain SJI1 and other closely related Desulfovibrio species.
50
Table 4.1 Force quantification of bacteria in artificial seawater on various metals 55
Table 4.2 Contact angle and surface charge of bacteria in artificial seawater 58
Table 4.3 Force quantification of three bacteria on SS316 in various solutions 66
Table 4.4 Force quantification of three bacteria on SS316 in ASW with different pH
71
Table 4.5 Fitting parameters for XPS spectra Fe2p3/2 and relative quantity of
compounds in the surface of SS316 immersed in ASW at different pH 73
Table 4.6 Tip-surface adhesion forces on coupons with a biofilm (mean ± S.D.) 78
Table 5.1 Parameters of EIS for the samples in EASW or EASW with SRB after 14
and 35 days of immersion 100
Table 6.1 Corrosion current analysis on the pristine SS316 and the functionalized
SS316 after immersion in EASW for 5 weeks 110
Table 7.1 Charge transfer resistance and corrosion inhibition efficiency parameters for
the corrosion of mild steel in EASW with or without MBI 118
Table 7.2 Electrochemical polarization parameters for pristine mild steel and inhibited
mild steel calculated from Tafel plots 121
Table 7.3 AFM study of biofilm surface roughness and pit depth 128
Table 7.4 Charge transfer resistance and corrosion inhibition efficiency parameters for
the corrosion of SS316 in EASW with or without MBI 132
Table 7.5 Electrochemical polarization parameters calculated from Tafel plots for the
pristine SS316 and the SS316 with MBI 134
Table 7.6 AFM study of biofilm surface roughness and pit depth 138

Nomenclature
xii
NOMENCLATURE

AFM Atomic force microscopy

ASW Artificial seawater

C Concentration of inhibitor

CPE Constant phase element

CLSM Confocal laser scanning microscope

DIW Deionized water

EASW Enriched artificial seawater

EIS Electrochemical impedance spectroscopy

EPS Extracellular polymeric substance

IE Inhibition efficiency

IOB/MOB Iron/manganese-oxidizing bacteria

MIC Microbiologically influenced corrosion

PCR Polymerase chain reaction

Q
b
Capacitance of the biofilm

Q
dl
Capacitance of the double layer

Q
f
Capacitance of the ferrous sulfide film

Q
pit
Capacitance of the pits in the passive film

Q
pf
Capacitance of the passive film

R
b
Resistance of the biofilm

R
ct
Charge transfer resistance

R
f

Resistance of the ferrous sulfide film

Nomenclature
xiii
R
s
Solution resistance

SEM Scanning electron microscope

SOB Sulfur-oxidizing bacteria

SPB Slime-producing bacteria

SRB Sulphate-reducing bacteria

SS316 Stainless steel 316

XPS X-ray photoelectron spectroscopy

Z
CPE
Impedance of the constant phase elements

i
corr
Corrosion current density

i' Corrosion current density of the inhibitor-containing mild steel

i Corrosion current density of the pristine mild steel

ρ
Specimen density

M Atomic mass of the metal

ΔG
o
ads


Free energy of adsorption

K
ad
Adsorption equilibrium constant

f Molecular interaction constant

θ
Surface coverage values

ω Angular frequency of alternating current voltage

d Cantilever deflection

k
sp
Cantilever spring constant

Chapter 1 Introduction
1
CHAPTER 1 INTRODUCTION
Although corrosion associated with microorganisms has been recognized for
over 50 years, research in biocorrosion (i.e. the role played by microorganisms in
corrosion) is considered relatively new and its mechanism is still not fully understood.
Biocorrosion, also termed microbiologically influenced corrosion (MIC), refers to the
influence of microorganisms on the kinetics of corrosion processes of metals, induced
by microorganisms adhering to the interfaces, i.e. on the biofilm.
Biocorrosion is not a new corrosion mechanism but it integrates the role of
microorganisms in the corrosion processes. It occurs directly and indirectly as a result
of the activities of living microorganisms. The corrosion reactions can be influenced
by microbial activities, especially when the microorganism attaches onto metal
surface to form biofilm. Kinetics of corrosion processes of metals can be influenced
by biofilms. Products of their metabolic activities including enzymes, exopolymers,
organic and inorganic acids, as well as volatile compounds such as ammonia or
hydrogen sulfide can affect cathodic and/or anodic reactions, thus altering the
electrochemistry at the biofilm/substrate interface. The involvement of biofilm on
metal surface may result in metal deterioration.
It is well-known that seawater is more corrosive than freshwater because of the
high concentration of chloride ion. Chloride can decrease the pH near the metal
surface and attack the passive film on the metals. Furthermore, seawater supports the
growth of diverse living microorganisms. When immersed in seawater, metal surfaces
Chapter 1 Introduction
2
are rapidly covered with a layer of primary bacterial film. The corrosion induced by
the microorganisms occurs after this bio-adhesion process.
Biofilm and biocorrosion have become a serious problem in the marine industry.

It reduces the lifetime of various industrial materials and equipment. It is estimated
that approximately 20% of all corrosion damage of metals is induced by biocorrosion
(Flemming, 1996). Financial cost associated with the repair and replacement of
equipment resulting from the damage of biofilm and biocorrosion problem run into
millions of dollars annually. Brennenstuhl et al. (1992) reported that biocorrosion
caused a damage of approximately US $ 55 million in stainless steel exchangers
within 8 years. The costs arise from lost energy, spare parts, repair efforts, monitoring
and changes in design.
Therefore, it is important to study the biocorrosion behavior of metals and its
corrosion mechanisms in the marine environment. There are usually several
mechanisms involved in biofilm induced corrosion. A biofilm not only entraps
deleterious metabolites secreted by bacteria, but also creates gradients of pH,
dissolved oxygen, nutrient, and chloride. Over time, this alters and influences the
immediate surroundings of the metal surface and leads to localized corrosion of the
metal.
The metabolic products of microorganisms in biofilm may be very harmful to
the metals. For example, the organic or inorganic acids produced by bacteria greatly
increase the corrosion of metals by speeding up the anodic reaction, while some
bacteria may be involved in the cathodic reaction by consumption of hydrogen or
Chapter 1 Introduction
3
oxygen (cathodic reactants) in the metal-biofilm interface. Therefore, it is important to
study the mechanism of the biofilm and biocorrosion. In this chapter, a review will be
given on biofilm formation, biocorrosion mechanisms, bacteria species associated
with the biofilm and biocorrosion of metals, as well as methods for the inhibition of
biofilm and biocorrosion.

1.1 Biofilm Formation on Metal Surfaces
Biofilm is composed of microorganisms (including bacteria, fungi, algae and
protozoa) adhering to the surfaces of solid in an aqueous environment. It is a slimy

substance which contains microorganisms, extracellular polymeric substances, metals,
plastics, and soil particles. Biofilm grows via a series of steps: First some trace
organics are first adsorbed to the surface to form a conditioning layer, after which
some pioneer bacteria may adsorb and subsequently desorb (Hamilton, 1987). The
initial bacteria attachment is formed through a reversible adsorption process, which is
governed by electrostatic attraction and physical forces, e.g. van der Waals forces and
hydrophobic interactions (Ong et al, 1999; Van Oss et al., 1986), but not
chemisorption. The adhesion forces are dependent on the physicochemical property of
the substrate and the surface property of bacteria, e.g. hydrophobicity and surface
charge. The initial bacterial attachment is a crucial step in the process of biofilm
development (Razatos et al., 1998).
Some researchers (Hamilton, 1987; Wolfaardt and Cloete, 1992) have taken an
empirical approach to observe initial microorganisms attachment microscopically, and
Chapter 1 Introduction
4
model the adhesion process. The attachment is usually studied by image analysis such
as confocal laser scanning microscopy (CLSM) developed by Caldwell and Lawrence
(1989). Some other researchers, including Absolom et al. (1983), and Rutter and
Vincent (1984), have expanded on the physicochemical thermodynamic approach.
Absolom et al. (1983) employed a concept of short-range interaction force to see the
direct bacteria contact with the substratum, and the Gibbs free energy is estimated
from the interfacial tension. In contrast, Rutter and Vincent (1984) used the
long-range interaction concept based on the DLVO (Derjaguin, Landau, Verway, and
Overbeek) theory. The interaction Gibbs free energy between particle and surface is a
function of the distance between the two. Recently, atomic force microscopy (AFM)
force measurements of cell-solid and cell-cell interactions using functionalized probes
have been shown to be a promising new approach to study the initial bacteria
attachment (Dufrêne, 2003). The bacteria are directly attached to the end of the
cantilever to form a modified tip (termed as a cell probe). Cell probes have been used
to quantify the interactions between the bacteria and various inanimate surfaces,

including mica, Teflon, some coated substrates (e.g. polystyrene), and hydrophilic as
well as hydrophobicly modified glass. It has been reported that cell adhesion to
surfaces is enhanced by the surface hydrophobicity of the substrate (Videla, 1996;
Ong et al., 1999). Lower et al. (2000; 20001a; 2001b) also used AFM force
measurements to quantify the interfacial and adhesion forces between bacteria and
mineral surfaces. Besides bacterial cells, cell probes that were modified with yeast
and spore have been employed for the analysis of fungal contamination in food, drug
Chapter 1 Introduction
5
and agricultural industries (Bowen et al., 2000a, 2000b, 2001, and 2002). Bowen et al.
(2000a, 2000b, 2001, and 2002) used different yeast cells and spores to study the
parameters that influence the cell adhesion, including the strength of cell-substrate
interactions, the time development of adhesive contact, the influence of pH and ionic
strength, the effect of substratum, and the effect of the culture age and growth
conditions. Interestingly, the cell probe can be used to “recognize” a mineral surface;
it has been reported that the affinity between the bacterium Shewanella oneidesis and
goethite rapidly increases as electrons transfer from the bacterium to the mineral
(Lower et al., 2001b).

AFM force measurements using a cell probe have also been
applied in the area of membrane research (Li and Elimelech, 2004; Hilal and Bowen,
2002; Hilal et al., 2003) to investigate the contamination and fouling of the
nanofiltration membrane. However, the bacterial attachment to metal surfaces has
seldom been studied.
In general, some of the adsorbed cells colonize and form structures which may
permanently hold the cells to the surface to form a biofilm. The adsorbed cells
produce extracellular polymeric substance (EPS), whether capsule or a loose network,
as a glycocalyx. Soon thereafter, a thriving colony of bacteria is established. In a
mature biofilm, more of the volume is occupied by the loosely organized glycocalyx
matrix (75% - 95%) than the bacteria cells (5 - 25%).

The development of biofilm is affected by some parameters (Coetser and Cloete,
2005) such as the system temperature, water flow rate past the surface, environmental
nutrient, surface roughness, and pH conditions of water which influence the bacterial
Chapter 1 Introduction
6
growth and attachment.

1.2 Mechanisms of Biocorrosion
The formation of biofilm may have deleterious effects for the metal substrates.
Two distinct classes of microorganisms, the aerobe and anaerobe, cause biocorrosion
with distinctly different types of corrosion reactions. Under aerobic conditions, the
continuous supply of oxygen to the cathode and the removal of the insoluble iron
oxides and hydroxides at the anode speed up the corrosion process (Hamilton, 1985).
The role of the microorganisms is either to assist in the establishment of the
electrolytic cell (indirect) or to simulate the anodic or cathodic reactions (direct)
(Hamilton, 1985).
The microorganisms in the biofilm increase the metal corrosion in several ways:
(a) Consumption of oxygen (cathodic reactant in aerobic corrosion) by aerobic
microorganisms to form localized differences in concentration shift, which results in
the creation of localized corrosion of metals.
(b) Consumption of hydrogen (cathodic reactant in anaerobic corrosion) by
microorganisms to depolarize the cathode, which increases the rate of metal loss at the
anode.
(c) Biodegradation of protective coatings on metal surfaces by microorganisms.
(d) Biodegradation of corrosion inhibitors, which are added to protect metals in
industrial water systems.
(e) Production of microbial metabolites which are corrosive organic and inorganic
Chapter 1 Introduction
7
acids, and are often the end-products of the metabolism of microorganisms.

(f) Production of metabolic by-products, such as H
2
S, which precipitate metal ions,
such as iron to form corrosive FeS.

1.3 Bacteria Related to Biofilm Formation and Biocorrosion
Microorganisms associated with biocorrosion of metals such as iron, aluminum,
copper and their alloys are diverse in the natural environment. Their ability to
influence the corrosion of metals by changing the corrosion resistance in the
environment makes the microorganisms deleterious to the metals.
The main types of bacteria involved in biocorrosion of metal substrates are (i)
sulphate-reducing bacteria (SRB), (ii) sulfur-oxidizing bacteria (SOB), (iii)
iron/manganese-oxidizing bacteria (IOB/MOB), and (iv) slime-producing bacteria
(SPB). These microorganisms can coexist in natural biofilms, and affect the
electrochemical processes in either anaerobic or aerobic reaction by the excreted
metabolites.
1.3.1 Sulphate-reducing Bacteria (SRB)
The most common bacteria related to biocorrosion are sulphate reducing
bacteria (SRB), which include the genus Desulfovibrio, Desulfotomaculum, and
Desulforomonas. SRB are anaerobes that are sustained by organic nutrients. Generally
they require a complete absence of oxygen and a highly reducing environment to
function efficiently. SRB are usually not the first group of microorganisms to deposit
on metals in the aqueous environment. Initially, aerobic microorganisms are the
Chapter 1 Introduction
8
predominant populations present in water. As these grow, biofilms accumulate and a
strong reducing environment develops at the attachment point. SRB then begin to
grow. The metabolites of the aerobic microorganisms not only produce reducing
conditions, but also provide nutrients for the SRB, which permit them to grow at a
rapid rate. Corrosion develops in the areas where SRB have grown to a high

population. Thus anaerobic biocorrosion occurs in aqueous systems. Although water
contains free oxygen, the areas where SRB grow are anaerobic.
The mechanisms of metal corrosion in the presence of SRB are complex. In an
anaerobic environment, SRB use sulphate as the electron acceptor and reduce it to
sulfide. Von Wolzogen Kuhr and van der Vlugt (1934) in their pioneering work,
suggested the following reactions occurring:
4Fe → 4Fe
2+
+ 8e
-
(anodic reaction)
8H
2
O → 8H
+
+ 8OH
-
(water dissociation)
8H
+
+ 8e
-
→ 8H(ads) (cathodic reaction)
SO
4
2-
+ 8H(ads) → S
2-
+ 4H
2

O (bacterial consumption)
Fe
2+
+ S
2-
→ FeS (corrosion products)
4Fe + SO
4
2-

+ 4H
2
O → 3Fe(OH)
2
+ FeS + 2OH
-
(overall reaction)
This overall process is described as cathodic depolarization. Based on this
theory, SRB consume the atomic or cathodic hydrogen which accumulates at the
cathode by a hydrogenase enzyme, thereby depolarizing the cathode (Hardy, 1983).
This is the first mechanism proposed for SRB induced corrosion.
Some researchers (Sanders and Hamilton, 1986; Little et al., 1992), however,
Chapter 1 Introduction
9
have suggested

that the corrosion rates increase due to the cathodic reduction of H
2
S:
H

2
S + 2e
-
→ H
2
+ S
2-
(cathodic reaction of H
2
S)
and the anodic reaction is accelerated by the formation of iron sulfide:
Fe + S
2-
→ FeS + 2e
-
(anodic reaction)
It is, however, generally acknowledged that it is too simplistic to consider only
one mechanism, since many factors may be involved in SRB-influenced corrosion.
Besides the cathodic depolarization by hydrogenase and anodic depolarization
demonstrated above, the corrosion process or substances involved may also include
iron sulfide, Fe-binding exopolymers, volatile phosphorus compound, sulfide-induced
stress corrosion cracking and hydrogen-induced cracking or blistering (Beech, 1999).
The three SRB induced corrosion mechanisms mentioned above are based on
the indirect interaction of SRB with metals, i.e. by increasing the anodic or cathodic
reaction. Recently, Dinh et al. (2004) detected SRB with the potential for direct
corrosion by enriching the SRB cultures with iron specimens as the only electron
donor and marine sediment as the inoculum. The growth of living bacteria suggests
that the SRB strain IS4 has a direct interaction with iron. An electron flow from
metallic iron can directly participate in the sulphate reduction via a pathway:
Fe electron transport system sulphate reduction enzymes

Hydrogenase H
2

Such direct interaction between SRB and metallic iron indicates that the iron
could become a growth substrate of SRB, which dramatically increases the metal
Chapter 1 Introduction
10
corrosion. This understanding greatly changes the conventional viewpoint toward
SRB induced corrosion, which is usually considered to be the result of the indirect
influence of SRB on the biocorrosion of metals.
These mechanisms mentioned above offer a possible explanation of
SRB-induced corrosion. However, several factors, such as the cathodic depolarization,
anodic depolarization, acidification caused by hydrogen sulfide, and the direct
electron flow between metal and bacteria, may influence biocorrosion of metals
simultaneously, thus rendering the biocorrosion behavior of SRB more complicated.
1.3.2 Other Bacteria
Besides SRB, numerous types of bacteria are able to carry out iron oxidizing
reactions and have been shown to influence corrosion reactions. Some bacteria
associated with the corrosion and their mechanisms are listed below:
(a) Iron/Manganese oxidizing bacteria (IOB/MOB)
IOB/MOB, for example, the genera Siderocapsa, Gallionella, Leptothrix,
Sphaerotilus, Crenothrix, and Clonothrix, are groups of bacteria related to MIC. They
can oxidize Fe
2+
, either dissolved in the bulk medium or precipitated on a surface, to
Fe
3+
. The dense accumulation of IOB/MOB on the metal surface may thus promote
the corrosion reactions by the deposition of cathodically reactive ferric and manganic

oxides and the local consumption of oxygen by bacterial respiration in the deposit
(Beech and Gaylarde, 1999). It has been shown that IOB/MOB can promote the
ennoblement of metals (i.e. a change to more positive values of pitting potential) and
pitting corrosion.